Tumor MHC Class I Expression Improves the ...

Published OnlineFirst February 20, 2014; DOI: 10.1158/2326-6066.CIR-13-0180

Cancer Immunology Research

Cancer Immunology Miniatures

Tumor MHC Class I Expression Improves the Prognostic Value of T-cell Density in Resected Colorectal Liver Metastases Simon Turcotte1, Steven C. Katz3, Jinru Shia2, William R. Jarnagin1, T. Peter Kingham1, Peter J. Allen1, Yuman Fong1, Michael I. D'Angelica1, and Ronald P. DeMatteo1

Abstract Tumor-infiltrating lymphocytes (TIL) in colorectal cancer liver metastases (CLM) have been associated with more favorable patient outcomes, but whether MHC class I (MHC-I) expression on cancer cells affects prognosis is uncertain. Immunohistochemistry was performed on a tissue microarray of 158 patients with CLM, who underwent partial hepatectomy with curative intent. Using the antibody HC-10, which detects HLA-B and HLA-C antigens and a minority of HLA-A antigens, MHC-I expression was correlated with b-2 microglobulin (b2m; r ¼ 0.7; P < 0.001), but not with T-cell density (r < 0.32). The median follow-up for survivors was 9.7 years. High levels of MHC-I expression in tumors concomitant with high T-cell infiltration (CD3, CD4, or CD8) best identified patients with favorable outcomes, compared with patients with one or none of these immune features. The median overall survival (OS) of patients with MHC-IhiCD3hi tumors (n ¼ 31) was 116 months compared with 40 months for the others (P ¼ 0.001), and the median time to recurrence (TTR) was not reached compared with 17 months (P ¼ 0.008). By multivariate analysis, MHChiCD3hi was associated with OS and TTR independent of the standard clinicopathologic variables. An immune score that combines MHC-I expression and TIL density may be a valuable prognostic tool in the treatment of patients with CLM. Cancer Immunol Res; 2(6); 530–7. 2014 AACR.

Introduction Individualizing the care of patients with metastatic colorectal cancer based on tumor biology requires biomarkers that estimate a patient's outcome better than what standard clinicopathologic variables accomplish currently. Accumulating evidence suggests that the adaptive immune system can influence cancer progression, and the quantification of tumor-infiltrating lymphocytes (TIL) may improve prognostic staging of patients with solid cancers (1). In this regard, primary colorectal cancer has been the most comprehensively studied tumor (2). A pivotal study of 406 patients with primary colorectal cancer showed that high intratumoral CD3þ T-cell density could identify patients with similar disease-free survival, independent of the depth of tumor penetration (T stage) or nodal metastases (N stage; ref. 3).

Authors' Affiliations: Departments of 1Surgery and 2Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York; and 3Department of Surgery, Boston University School of Medicine, Roger Williams Medical Center, Providence, Rhode Island Note: Supplementary data for this article are available at Cancer Immunology Research Online (http://cancerimmunolres.aacrjournals.org/). de Montre al, Centre Corresponding Author: Simon Turcotte, Universite de Montre al (CRde Recherche du Centre Hospitalier de l'Universite CHUM), 900, rue Saint-Denis (Tour Viger), Room R10.430, Montreal, QC H2X 0A9, Canada. Phone: 514-890-8000, ext. 35328; Fax: 514-412-7480; E-mail: [email protected] doi: 10.1158/2326-6066.CIR-13-0180 2014 American Association for Cancer Research.

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In addition, the intratumoral T-cell density and quality have been inversely correlated with colorectal cancer progression (4, 5). For instance, the primary tumors of patients with metastatic colorectal cancer to distant organs [tumor–node–metastasis (TNM) stage IV; n ¼ 86] harbored two to three times fewer CD8þ T cells and three to five times fewer granzyme Bþ T cells than tumors of patients with only regional lymph node metastases (TNM stage I to III; n ¼ 312; ref. 5). Although these findings suggest that metastatic tumor deposits represent immune escape variants, we and others have shown that TIL in colorectal cancer liver metastases (CLM) had a prognostic value after complete resection (6, 7) or chemotherapy (8). When considered alone, however, TIL density in CLM seems to be only a modest predictor of clinical outcomes. Partial or total loss of MHC class I (MHC-I) expression is regarded as a common tumor immune escape mechanism, which theoretically can render cancer cells "invisible" to CD8þ T cells. MHC-I loss has been reported at high frequency in solid tumors (9) and in up to 74% of primary colorectal cancers (10). Conversely, the HLA and b-2 microglobulin (b2m) genes, encoding the MHC constituents, are IFN responsive and their expression can be upregulated in a tumor microenvironment in which productive immune recognition occurs. The prognostic value of MHC-I expression is uncertain in primary colorectal cancer (11, 12), but strong MHC-I tumor expression combined with high CD3 TIL density has been associated with modestly longer disease-specific survival compared with patients with either featurealone(72.5,68.0,and69.9months,respectively;refs.11,13).

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MHC Class I and T Cells in Colorectal Liver Metastases

The aim of this study was to analyze whether prognostic immune scoring in metastatic colorectal cancer could be improved by assessing MHC-I expression in conjunction with TIL quantification in CLM resected with curative intent.

Patients and Methods Patients We identified from a prospective database consecutive patients who underwent resection of CLM with curative intent at our institution between 1998 and 2000 (7). Indications for resectability have been described previously (7, 14). Institutional Review Board approval was obtained. We previously developed a clinical risk score (14), which estimates postoperative outcome and has been validated by others (15). To calculate the clinical risk score, a point is given for each of the following clinicopathologic characteristics: node-positive primary cancer, disease-free interval (DFI; time between resection of primary and liver recurrence) 5 cm, and prehepatectomy serum carcinoembryonic antigen (CEA) level >200 ng/mL. Immunohistochemistry Following pathologic review for diagnostic confirmation and exclusion of highly fibrotic or necrotic tumors, tissue microarrays (TMA) were constructed from 188 patients as described previously (7). Cores measuring 0.6 mm in diameter were made in triplicate from paraffin blocks and processed using the ATA27 automated arrayer (Beecher Instruments). TMA blocks were cut to 5-mm sections, deparaffinized, rehydrated in graded alcohol, and stained with biotinylated secondary antibodies and positive or isotype controls. CD3, CD4, CD8, and Fox3 staining and quantification have been reported separately (7). We used a validated mouse anti-human monoclonal antibody that binds to MHC-I heavy chains, preferentially for the HLA-B and HLA-C molecules, and seven HLA-As (HC-10; provided by Hidde L. Ploegh, Whitehead Institute, Cambridge, MA; 1:1,000; 1 hour; refs. 16, 17). The polyclonal rabbit anti-human antibody reacting to light-chain b2m was used (A0072; DAKO; 1:50,000; 1 hour). Automated staining was done on a Ventana XT with the OmniMap DAB Detection System (Roche). Nuclei were counterstained with hematoxylin. High-resolution TMA digital images were acquired on a MIRAX SCAN (Carl Zeiss) and quantification was carried out with the Metamorph Image Analysis Software (Molecular Devices) blinded to clinical data. The areas of positive signal and the total area of the tissue core were calculated on the basis of color, where pixels with identical RGB (red, green, and blue) values were grouped together, to calculate a ratio of positive brown staining (moderate to strong) over total staining (all brown and hematoxylin blue) for each core (Supplementary Fig. S1). Thresholds were set to avoid connective tissue, fat, and necrosis. Mean SE was calculated per tumor replicate. Quantification of MHC-I on full cores was compared with quantification on zones of tumors excluding stromal bands and necrotic areas, and found to be similar and highly correlated (Spearman r ¼ 0.993; P < 0.001; Supplementary Table S1). Patients were excluded from the analysis when at least one tumor core could not be quantified for MHC-I expression.

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Statistical analysis Patient disease status was updated through April 2013. Overall survival (OS) and time-to-recurrence (TTR) were calculated from the time of hepatectomy by the Kaplan–Meier method. Groups were compared by the log-rank test. The association between immune parameters and outcome was also evaluated by univariate Cox regression on continuous variables, and using optimal cutoff points selected by the maximally selected c2 method (R version 2.7; www.r-project. org) to estimate the best separation between groups with P values corrected for overfitting (7, 18). Forward selection stepwise multivariate Cox regression models were applied to the MHChiCD3hi immune score and significant clinicopathologic prognostic factors. The Spearman r test was used to assess the correlation between continuous variables, and the Pearson x2 test for the association between categorical variables. A two-sided P value of 0.05 was considered statistically significant (SPSS version 21).

Results Clinicopathologic features For this study, we have identified 188 patients with CLM, who underwent partial hepatectomy with curative intent at our institution; samples from 158 of these patients were analyzed after those from 20 patients were excluded for lack of quantifiable cores, 8 for inadequate follow-up, 1 for palliative resection, and 1 for duplicative sample. The median age at hepatectomy was 63 years, and 57% were male. The median follow-up time was 42 months overall and 116 (41–171) months for survivors. At the last follow-up, there were 35 patients (22%) alive without disease, 106 patients (67%) had cancer recurrence, and of the 114 patients (72%) that died 84% of the deaths were from the cancer. The median OS was 46 months. The 5- and 10-year predicted survival rates were 39% and 24%, respectively. The median TTR was 20.4 months. The 5- and 10year predicted recurrence-free survival rates were 30% and 25%, respectively. Clinicopathologic variables significantly associated with longer OS and TTR (Table 1) were resection margins clear of cancer, and most components of the clinical risk score (14). By these conventional criteria, the longest median OS and TTR were 77.4 and 31.4 months for the 53 patients (34.6%) with the lowest clinical risk scores (0 or 1). Immunologic features MHC-I and b2m expressions in CLM were quantified to obtain the percentage of expression per tumor core (Supplementary Fig. S1). The distribution of MHC-I expression across CLM ranged uniformly from undetectable to high levels (range, 1.4%–92.3%; median, 47.7%; terciles, 32.1% and 65.0%; Fig. 1A). This broad distribution allowed the use of terciles as cutoff points to group patients by null/low, moderate, or high MHC-I expression level. The specificity of MHC-I detection was supported by strong correlation with b2m expression (r ¼ 0.69; P < 0.001); however, the b2m distribution was skewed toward the lower values (range, 1.0%–84.8%; median, 20.9%; terciles, 13.1% and 32.7%; Fig. 1A). The imperfect correlation between MHC-I and b2m was consistent with the preferential binding of HC-10 to HLA-B and HLA-C heavy chains free of b2m (16, 17).

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Turcotte et al.

Table 1. Univariate analysis of clinicopathologic and immune characteristics for survival and recurrence OS Variables Clinicopathologic Age, y Gender Site of primary cancer Perioperative chemotherapy Major resection (3 segments) Cancer at resection margin No. of hepatic metastasesa,b Size of largest tumor, cma Preoperative CEA, ng/mLa Node-positive primarya DFI, moa Clinical risk score

Immune featuresc MHC-I b2m CD3 CD4 CD8 Combination of immune features MHC-IhiCD3hi MHC-IhiCD4hi MHC-IhiCD8hi

TTR

Cutoff

n

(%)

>63 63 Male Female Colon Rectum Yes No No Yes Negative Positive Solitary >1 5 >5 200 >200 No Yes 12